As the most common bacterial infection in childhood, otitis media is associated with enormous human and economic consequences. A large subset of children (~10% of all children) experience recurrent and chronic otitis media, exacting a huge toll on children and their families. These otitis prone children would benefit most from better treatment and prevention strategies. A critical first step in the pathogenesis of otitis media is colonization of the nasopharynx (NP) by one or more of the 3 primary bacterial pathogens of otitis media: nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae and Moraxella catarrhalis. Given the critical role of NP colonization in the pathogenesis otitis media, one might surmise that the administration of chronic prophylactic antibiotics to otitis prone children to prevent NP colonization would be a viable option. Unfortunately this approach is fraught with problems. Antibiotics eradicate the normal protective flora, select antibiotic resistance in pathogens and commensals and are associated substantial morbidity. Prophylactic antibiotics are simply not a viable option for most otitis prone children. We propose an entirely novel strategy to intervene at the step of NP colonization using antisense molecules to eradicate otitis media pathogens specifically and leave the normal flora intact. The development of antisense molecules as therapeutic agents has opened a potentially new era in antimicrobial therapy. The overall concept is to target sequence-specific genes that are important for viability, eradicating the pathogen but leaving normal flora undisturbed. We have developed preliminary studies to show that specific peptide nucleic acid antisense molecules conjugated to membrane-penetrating peptides have potent antimicrobial activity against NTHI in vitro. Looking ahead, we would envision that otitis prone children would be candidates for a strategy of specific eradication of pathogens using topical administration of such compounds, for example using nose drops or nasal sprays. In aim 1 we will assess in vitro antimicrobial activity of antisense molecules that target specific genes that are important for viability of bacterial otitis media pathogens. In aim 2 we will examine antisense molecules in preventing and disrupting biofilms. In Aim 3 we will evaluate selected antisense molecules to kill intracellular bacteria in respirator epithelial cells and macrophages, cells known to be reservoirs of otitis media pathogens in the human upper respiratory tract. In spite of substantial advances in our understanding of the pathogenesis, etiology, genetics and biology of otitis media, no truly new treatment modalities for infection in this setting have been introduced in decades. The development of specific antisense molecules represents a potentially new era of antibiotic therapy in the setting of otitis media (and many other infections) to specifically eradicate a pathogen without perturbing the normal flora.